The use of viral vectors for cell reprogramming is a powerful approach for translational medicine. Promising results have been obtained with the use of viral transduction in stem cell research (Okita, et al., (2010) Exp Cell Res 316 (16):2565-2570. doi:10.1016/j.yexcr.2010.04.023), transplantation (Ritter, et al., (2009) Curr Opin Mol Ther 11 (5):504-512), and immunotherapy (Collins, et al., (2008) Curr Gene Ther 8 (2):66-78). Recent clinical trial results using this approach to reprogram T cells against lymphomas are encouraging (Kay (2011) Nature Reviews Genetics 12 (5):316-328). Still, the introduction of viral genomes into human cells leads to some negative consequences, among them: (a) genomic integration of the viral vectors, leading to insertional mutagenesis, and potential transformation of the host cells (Hacein-Bey-Abina, et al., (2003) Science (New York, N.Y.) 302 (5644):415-419); (b) long or permanent presence of reprogrammed cells in the body that increase the burden on immune system and can lead to distant complications after completion of the treatment (Porter, et al., (2011) New England Journal Medicine 365 (8):725-733); (c) continuing production of viral proteins that could induce host immune responses.
The use of non-integrated viral vectors can reduce the risk of insertional mutagenesis; but this does not address the problem of long-term persistence of vector and raises concerns of uncontrolled viral modification in host cells.
Transfection with synthetic mRNA is an important method of cell reprogramming that makes it possible to avoid the abovementioned problems (Rabinovich et al., 2009, Rabinovich P M, Weissman S M (2012) Cell engineering with synthetic messenger RNA Synthetic Messenger RNA and Metabolism Modulation Methods in Molecular Biology 969 (In Press)). mRNA introduced into cells exists only in the cytoplasm and does not cause genome perturbations. mRNA mediated reprogramming is essentially transient. Unless expression of the mRNA changes the cell epigenetically, transient transfection is limited by the time of mRNA and cognate protein persistence in the cell, and does not continue after degradation of cognate proteins.
Despite its attractive features, present technologies for mRNA transfection are hampered by the relatively rapid degradation of mRNA. In general, mRNA half-life is short and often is in the range of 2-5 h. Short mRNA persistence makes the duration of its expression mainly a function of the cognate protein stability. For example, the duration of expression of stable proteins can last weeks. Alternatively, the unstable protein HOXB4 can be detected for only a few hours after transfection of its encoding mRNA. Similarly, secretory proteins, excreted from cells soon after translation from transfected mRNA, are generally detected in the cells only for a few hours after transfection. Some methods have been employed to increase mRNA stability, for example, protection of mRNA termini from exonuclease degradation (Holtkamp, et al., (2006). Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108 (13):4009-4017) (Grudzien-Nogalska, et al., (2007) RNA 13 (10):1745-1755) (Rabinovich, et al., (2006) Hum Gene Ther 17 (10):1027-1035), however these methods alone are generally insufficient to substantially increase the duration of expression of cognate proteins in cells.
Another approach to cell reprogramming includes the use of negative strand RNA viruses that can form highly stable cytoplasmic ribonucleoprotein structures with a long lasting ability for mRNA and protein synthesis (Biology of Negative Strand RNA Viruses: The Power of Reverse Genetics (2010)). However, the use of viral RNA to produce viral vectors is hampered by the inability of the naked viral RNA to efficiently initiate viral transcription or replication. Attempts to address this problem include the use various DNAs encoding viral vector, viral nucleocapsid protein (NP) and the replicase/transcriptase complex proteins P and L to rescue the viral genome RNA. NP encapsidates viral RNA in a nucleoprotein complex, and makes the RNA highly resistant to nuclease degradation; and P and L form PL, a complex that recognizes NP-encapsidated viral RNA and carries out at least two enzymatic activities: NP-RNA dependent RNA polymerase and NP-RNA dependent transcriptase (Walpita, et al., (2005). FEMS Microbiol Lett 244 (1):9-18); (Bitzer, et al., (2003) J Gene Med 5 (7):543-553).
The standard Sendai vector rescue system includes a plasmid coding viral RNA expression cassette under a T7 promoter, and 3 supplemental plasmids coding NP, P and L protein, also under T7 promoters. These four plasmids can be introduced in cells to produce T7 polymerase to produce viral vector RNA and NP, P and L proteins (Bitzer, et al., (2003) J Gene Med 5 (7):543-553). The rescue occurs with very low efficiency, usually <10−6, however, following rescue, the viral vector can replicate and produce viral particles sufficient for further rounds of transduction. In an alternative design, the rescue can be provided in cells which contain viral packaging proteins, namely F, M and NH (Yoshizaki, et al., (2006) J Gene Med 8 (9):1151-1159). In this case viral particles would contain defective virus, not able to produce complete viral particles outside of the packaging cells. In both cases, targeted cells obtained virus that continued to produce viral proteins such as N, P, and L and was able to replicate itself or, potentially, even to recover some level of virulency as result of natural propagation and selection.
Accordingly, it is an object of this invention to provide compositions and methods for cell reprogramming with improved safety and efficacy.
It is also an object of the invention to provide compositions and methods for prolonged recombinant protein stability and expression in cells via transient transfection of mRNA.